Mobilizing endogenous progenitor cells in the adult brain for treatment of neurodegenerative and demyelinating disease offers several advantages over transplantation of stem cells (SCs). The present project is investigating the ability of the cytokine leukemia inhibitory factor (LIF) to stimulate SC and progenitor cell production, and to direct their differentiated fates in a demyelination model. Since LIF is up- regulated in response to a variety of human brain injuries and diseases, experimentally increasing the level of LIF in the adult brain serves to enhance the normal response mechanism. Elevating LIF in the adult mouse brain promotes the self-renewal of NSCs, thereby increasing the number of cells that may be available for repair. In addition, LIF stimulates oligodendrocyte progenitor cell (OPC) proliferation and promotes oligodendrocyte survival. Thus, LIF can enlarge the NSC pool as well as direct NSCs towards fates that are of potential clinical importance. (1) It is proposed to examine the efficacy of this cytokine in the cuprizone model of demyelination, in which the onset of de- and remyelination can be experimentally controlled. Preliminary results indicate that exogenous LIF can significantly increase the number of OPCs and oligodendrocytes following demyelination. (2) Preliminary results also indicate that remyelination is strongly improved by LIF treatment. To explore the possibility of LIF-driven functional recovery, a collaboration has been established to study axonal conduction. (3) How does LIF increase OPCs, oligodendrocytes and myelin? Does it act directly on each of these cell types to direct their proliferation and differentiation? Preliminary results suggest that LIF acts directly on both newly generated OPCs and oligodendrocytes. Since LIF also activates glial cells, which in turn may influence OPCs, we will ask whether LIF stimulates OPC proliferation by direct signaling through its receptor in OPCs by conditionally deleting the gp130 component of the LIF receptor in OPCs. Revised Specific Aims (modified for 2-year funding) Mobilizing endogenous progenitor cells in the adult brain for treatment of neurodegenerative and demyelinating disease offers several advantages over transplantation of embryonic or neural stem cells (NSCs). We are investigating the ability of the cytokine leukemia inhibitory factor (LIF) to stimulate stem and progenitor cell production, and to help direct their differentiated fates following demyelination. Since LIF is up-regulated in response to a variety of human brain injuries and diseases, increasing the level of LIF in the adult brain by injecting the recombinant protein, or viral vectors encoding it, serves to enhance the normal response mechanism. During the prior granting period, we found that elevating LIF promotes the self-renewal of NSCs in the adult brain subventricular zone (SVZ), thereby increasing the number of cells that may be available for repair. In addition, we showed that LIF stimulates oligodendrocyte progenitor cell (OPC) proliferation in the brain, and promotes oligodendrocyte survival following spinal cord injury. Finally, we found that LIF enhances the replacement of injured adult olfactory sensory neurons. Thus, LIF has the ability to not only enlarge the NSC pool, but also to direct neural stem and progenitor cells towards fates that are of potential clinical importance. We propose to explore these properties of LIF a mouse demyelination model. A1. Test the ability of LIF to increase OPCs and oligodendrocytes following demyelination. The congenital and acquired CNS demyelinating diseases of children and adults are attractive targets for cell replacement therapy because the primary cell loss is of a single cell type, the oligodendrocyte. We found that exogenous LIF increases PHS 398/2590 (Rev. 11/07) Page 7 Continuation Format Page Patterson, Paul H the number of OPCs in the normal mouse brain as well as mature oligodendrocytes in injured spinal cord. Therefore, we are examining the efficacy of this cytokine in the cuprizone model of demyelination, in which the onset of de- and remyelination can be experimentally controlled to determine whether LIF can promote oligodendrocyte generation in the context of demyelination. We will deliver LIF protein or a viral vector encoding LIF and examine OPC proliferation, as well as the number of newly generated, mature oligodendrocytes using cell stage-specific markers coupled with BrdU administration. Preliminary results indicate that exogenous LIF significantly increases the number of OPCs and oligodendrocytes following cuprizone-induced demyelination. Hypothesis: Delivery of LIF can stimulate OPCs and restore oligodendrocyte numbers to normal levels following demyelination. A2. Test the efficacy of LIF to support remyelination and restore function. In light of these encouraging preliminary results, we have begun to examine remyelination in the cuprizone model. First, we are quantifying the extent of myelination using immunohistochemistry for myelin proteins. Second, we are assessing recovery of the characteristic features of nodes of Ranvier by assaying for markers of nodal, paranodal, and juxtaparanodal structures. Third, we are using electron microscopy (EM) to examine the ultrastructural features of the newly formed myelin and to quantify myelin thickness and the percent myelinated fibers in the medial corpus callosum (CC). To explore the possibility of LIF-driven functional recovery, we have established a collaboration to study axonal conduction using electrophysiology. Hypothesis: LIF can stimulate remyelination and aid in functional recovery in the cuprizone demyelination model. A3. Determine the targets of exogenous LIF action and the range of its effects on those targets. How does LIF stimulate OPC proliferation, oligodendrocyte production and myelin formation? Does it act directly on OPCs to direct their proliferation and their differentiation? Or does LIF set in motion a train of events that is subsequently controlled by other factors? In the in vivo setting, we are first characterizing the progenitor cells that proliferate in response to LIF by combining BrdU and cell- and stage-specific markers with staining for phosphorylated STAT3 (pSTAT3), a reliable marker for detecting LIF activation of the signal cascade downstream of its receptor. Preliminary results suggest that LIF acts directly on both newly generated OPCs and oligodendrocytes. Second, since LIF also activates microglial cells and astrocytes, which in turn may influence OPCs, we will ask whether LIF stimulates OPC proliferation by direct signaling through its receptor in OPCs by conditionally deleting the gp130 component of the LIF receptor in OPCs. Hypothesis: LIF stimulates oligodendrocyte generation directly by activating gp130 signaling in OPCs. To facilitate completion of our aims in a two-year funding period, we have eliminated several non-essential experiments from Aim 1 and Aim 3. These changes are outlined below. Aim 1 modifications The majority of the experiments in this proposal take advantage of the cuprizone model, which is the best model for our proof-of-concept experiments. In our most recent proposal, we included an additional experiment using a chronic EAE demyelination model (D1, Expt. 3), the aim of which was to test the efficacy of LIF in stimulating OPC proliferation and oligodendrocyte generation in a model that more closely mimics the pathology observed in MS. While understanding whether the effects of LIF that we observe in the cuprizone model extend to an inflammatory demyelination model is important for an evaluation of the therapeutic potential of LIF for human demyelinating PHS 398/2590 (Rev. 11/07) Page 8 Continuation Format Page Patterson, Paul H disease, it is not necessary for our initial proof-of-concept experiment, and thus this experiment can be delayed until further funding is obtained. Aim 2 modifications We have made no modifications to this aim as we think these experiments are critical for our proof-of-concept experiments. Furthermore, our encouraging preliminary results, including both those reported in the progress report and those accumulated since submission, make us confident that we can complete all components of this aim within two years. Aim 3 modifications Two of the reviewers stated that our third aim was our most interesting aim, and we agree, but nevertheless several of the experiments in this aim are not essential for our initial proof-of-concept study since they shift the focus from the initial characterization of the potential therapeutic value of LIF delivery following demyelination to an exploration of the mechanism underlying LIF actions. First, we have eliminated the experiment in which we conditionally delete gp130 in PDGFRa+ OPCs following cuprizone treatment (D3, Expt. 2b). We still intend to conditionally delete gp130 in OPCs just prior to LIF treatment, which will be straightforward and will address our primary question: does LIF stimulate OPC proliferation directly through the activation of gp130 signaling in a cell-autonomous manner? Second, we have eliminated our experiment aimed at understanding whether the continued presence of LIF has additional effects on differentiating oligodendrocytes beyond the stimulation of OPC proliferation (D3, Expt. 3). We initially suggested that this experiment would be completed in year 4, and it is not currently high on our priority list. Eventually, it will be necessary know whether transient LIF treatment is sufficient for its therapeutic effects or whether long-term LIF delivery is required, but these experiments can be delayed until further funding is obtained. Finally, we have eliminated the analysis of demyelination and remyelination in LIF KO mice. While our preliminary findings of delayed oligodendrocyte generation are intriguing, demonstrating whether the cause of this delay stems from a reduced progenitor response, a decrease in survival of the newly generated oligodendrocytes, or any one of several other potential causes, which we outlined in our proposal (D3, Expt. 4), will involve the assessment of LIF KO and WT mice at multiple additional time points during cuprizone treatment. This would require a significant number of experimental animals and extensive tissue processing, immunostaining, microscopy and analysis. Therefore, without further assistance, completion of this experiment would be difficult. Removal of this aim does not undermine our overall objectives.